www.nature.com/scientificreports OPEN Characterization of zebrafsh GABAA receptor subunits Kenichiro Sadamitsu, Leona Shigemitsu, Marina Suzuki, Daishi Ito, Makoto Kashima & Hiromi Hirata* γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, exerts its efect through the activation of GABA receptors. GABAA receptors are ligand-gated chloride channels composed of fve subunit proteins. Mammals have 19 diferent GABAA receptor subunits (α1–6, β1–3, γ1–3, δ, ε, π, θ, and ρ1–3), the physiological properties of which have been assayed by electrophysiology. However, the evolutionary conservation of the physiological characteristics of diverged GABAA receptor subunits remains unclear. Zebrafsh have 23 subunits (α1, α2a, α2b, α3–5, α6a, α6b, β1–4, γ1–3, δ, π, ζ, ρ1, ρ2a, ρ2b, ρ3a, and ρ3b), but the electrophysiological properties of these subunits have not been explored. In this study, we cloned the coding sequences for zebrafsh GABAA receptor subunits and investigated their expression patterns in larval zebrafsh by whole- mount in situ hybridization. We also performed electrophysiological recordings of GABA-evoked currents from Xenopus oocytes injected with one or multiple zebrafsh GABAA receptor subunit cRNAs and calculated the half-maximal efective concentrations (EC50s) for each. Our results revealed the spatial expressions and electrophysiological GABA sensitivities of zebrafsh GABAA receptors, suggesting that the properties of GABAA receptor subunits are conserved among vertebrates. γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system of vertebrates, 1 controls the excitability of neural networks mainly through GABA A receptors . Te GABAA receptor mediates two types of inhibition, known as phasic and tonic inhibition2. Phasic inhibition occurs at postsynaptic sites, where the GABA concentration transiently rises to more than 1 mM during synaptic transmission 3, while tonic inhibition occurs at extrasynaptic sites, where the concentration of spillover GABA increases to ~ 0.5 µM4,5. – Regardless of synaptic or extrasynaptic sites, GABAA receptors comprise fve subunits forming Cl conducting channels. Each subunit has a large extracellular N-terminal domain that contributes to GABA binding, followed by four transmembrane domains, with the second one forming the channel pore. Mammals have 19 difer- ent GABAA receptor subunits (α1–6, β1–3, γ1–3, δ, ε, π, θ, and ρ1–3). Although this diversity may allow for numerous possible combinations of subunits, most GABAA receptors are composed of two α, two β, and one γ 6 subunit . In fact, experimental evidence of native GABAA receptors suggests that there are fewer than 20 receptor 7,8 subtypes, with the major synaptic GABAA receptor combinations being α1β2γ2, α1β3γ2, α2β3γ2, and α3β3γ2 . Te extrasynaptic GABA A receptors appear to contain specifc subunits such as α4, α5, α6, and δ, forming α4β3δ, α5β3γ2, and α6β3δ2,7. Te other subunits—ε, π, and θ—also assemble with α and β subunits and are located at 2 extrasynaptic sites . Te ρ subunits form homopentameric GABA A receptors that are predominantly expressed in the retina9. Te β3 subunit can also form homopentameric channels that are activated by the anesthetic agent etomidate and a high concentration (~ 10 mM) of GABA in Xenopus oocytes10. While the physiological properties of GABAA receptor isoforms in mammals have been addressed, the evolutionary conservation and physiological signifcance of diverged subunits in vertebrates remain largely unclear. GABAA receptors have also been studied in zebrafsh, a vertebrate model that ofers advantages such as the production of many ofspring, fast embryonic development, optical transparency during embryogenesis, rapid acquisition of locomotor behaviors, and the ease of pharmacological treatment 11. Cocco and colleagues identifed 23 putative GABAA receptor subunits (eight α, four β, three γ, one δ, one π, one ζ, and fve ρ) in the zebrafsh 12 genome . Tey also investigated the transcript levels of GABAA receptor subunits in the adult zebrafsh brain using reverse transcription-polymerase chain reaction (RT-qPCR). Another recent study assayed the spatial expression patterns of eight GABA A receptor α subunits in zebrafsh embryos by in situ hybridization and showed that most α subunits are expressed during embryogenesis13. Several loss-of-function studies have revealed the physiological function of GABAA receptors in zebrafsh. Antisense morpholino-mediated knockdown of the α1 subunit caused reduced spontaneous locomotor activity in larvae at 5 days post-fertilization (dpf)14, while CRISPR/Cas9-mediated knockout of α1 caused seizure phenotypes in juveniles at 35 dpf15. Knockdown of the Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, Sagamihara 252-5258, Japan. *email: [email protected] Scientifc Reports | (2021) 11:6242 | https://doi.org/10.1038/s41598-021-84646-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ α2 subunit perturbed the expression of the proneural gene neurod and a GABA-synthesizing enzyme gene gad1b within 1 day of development 16. Zebrafsh larvae lacking the β3 subunit showed reduced sensitivity to 17 anesthetic drugs such as etomidate and propofol . Patch-clamp recordings of GABAA receptor-mediated min- iature inhibitory postsynaptic currents from zebrafsh Mauthner cells revealed three diferent types of gating kinetics, suggesting that zebrafsh also have multiple GABA A receptor subtypes comprising diferent subunit 18 combinations . However, the electrophysiological characteristics of the zebrafsh GABAA receptor subunit have not yet been explored. In this study, we performed phylogenetic analysis and cloned cDNAs for zebrafsh GABA A receptor subunits. Our whole-mount in situ hybridization revealed the spatial expression patterns of GABA A receptor subunit genes in 5 dpf larvae. We also assayed GABA-mediated gating of zebrafsh GABAA receptors composed of vari- ous combinations of receptor subunits in Xenopus oocytes. Tese attempts provide useful information on the spatial expressions and electrophysiological GABA sensitivities of zebrafsh GABAA receptors and suggest that the properties of GABAA receptor subunits are conserved among vertebrates. Results Phylogenetic analysis and cloning of zebrafsh GABAA receptor subunits. Nineteen GABAA receptor subunits/genes have been identifed in mammals (α1–6/gabra1–6, β1–3/gabrb1–3, γ1–3/gabrg1–3, 19 δ/gabrd, ε/gabre, π/gabrp, θ/gabrq, and ρ1–3/gabrr1–3) . Previous searches for GABAA receptor subunits in the zebrafsh genome database have suggested 23 GABA A receptor subunits/genes comprising eight α (α1/gabra1, α2a/gabra2a, α2b/gabra2b, α3–5/gabra3–5, α6a/gabra6a, and α6b/gabra6b), four β (β1–4/gabrb1–4), three γ (γ1–3/gabrg1–3), one δ/gabrd, one π /gabrp, and fve ρ (ρ1/gabrr1, ρ2a/gabrr2a, ρ2b/gabrr2b, ρ3a/gabrr3a, ρ3b/gabrr3b) as well as additional ζ/gabrz subunits, but neither ε nor θ subunits 12,13. Some subunits that have a or b at the end of the subunit/gene name are paralogs generated by a suspected duplication of the whole 20 genome during fsh evolution . We recapitulated in silico analysis using human and mouse GABAA receptor protein sequences as queries to obtain zebrafsh GABAA receptor protein sequences. We successfully cloned cDNAs for all zebrafsh GABAA receptor subunits except for α2b from an RNA mixture extracted from a pool of 1–5 dpf zebrafsh embryos/larvae. Te previously annotated zebrafsh α2b subunit (XP_017214538.1) showed 86% amino acid identity to the zebrafsh α2a subunit in the N-terminus (exons 1–8) and only 10% identity in the C-terminus (exon 9). Terefore, the α2b information has been removed from the National Center for Bio- technology Information (NCBI) annotation as it was presumably caused by an incorrect annotation of the last exon. We then searched for another exon encoding the C-terminus of α2b in the genome database using the C-terminus protein sequence of zebrafsh α2a as a query and identifed the other last exon encoding a possible α2b C-terminus that showed 76% amino acid identity to zebrafsh α2a. We successfully cloned the intact coding sequence of this newly annotated subunit and named zebrafsh GABAA receptor α2b subunit (LC596832), which difered from the previous annotation only in the last exon. We then updated the phylogenetic tree of human, mouse, and zebrafsh GABA A receptor subunits (Fig. 1). Our amino acid alignments of the GABA A receptor subunits showed that each subunit is conserved among vertebrates, especially in four transmembrane domains (Supplementary Figs. 1–17; Supplementary Table 1). We also confrmed that the ζ subunit, which is found in zebrafsh but not in mammals, belongs to the π subfamily, with the highest similarity to the zebrafsh π subunit, indicating that the ζ subunit is a paralog of the π subunit. Tus, we suggest renaming the π and ζ subunits to πa and πb subunits, respectively. We hereafer refer to π and ζ as π/πa and ζ/πb, respectively. Spatial expression of zebrafsh GABAA receptor genes. A previous RT-PCR analysis described the 12 expression of some, but not all, GABAA receptor genes in the brain and eye of adult zebrafsh . Another whole- mount in situ hybridization study reported the spatial expression patterns of eight subunit genes in zebrafsh embryos at 1, 2, and 4 dpf13. Since zebrafsh larvae with a defect in the α1 gene showed seizure-like motor activ- 15 21 ity as early as 4 dpf and GABAA receptor antagonist-induced